Cytokeratin Immunostaining Research Articles

Carcinoma neuroendócrino de pequenas células

O presente estudo reporta o caso de uma mulher de 63 anos da qual a única informação clínica era a suspeita de um sarcoma da cérvix. Simultaneamente à colpocitologia, foram enviadas biópsias do colo e do endométrio para diagnóstico. A visualização da amostra citológica revelou vários agregados de número variável de células monótonas, com tamanho pequeno, formato redondo e citoplasma escasso, num fundo com diátese. Os núcleos apresentavam moldagem, hipercromasia, cromatina “sal-e-pimenta” e ausência de nucléolos. O aspeto microscópico das biópsias foi concordante com os achados citológicos, tendo sido igualmente identificados focos glanduliformes com características atípicas. A neoplasia mostrou expressão imunohistoquímica dos antigénios enolase neurónio-específica (neuron specific enolase, NSE), sinaptofisina e citoqueratina (clones AE1/AE3), e uma elevada atividade proliferativa demonstrada pela imunorreactividade para o marcador nuclear Ki67/Mib1. Os achados citológicos, histológicos e imunohistoquímicos foram consistentes com o diagnóstico de carcinoma neuroendócrino de pequenas células. Dos tumores cervicais, esta neoplasia maligna é das mais raras, mostrando um comportamento muito agressivo, com prognóstico muito pobre, em que as terapêuticas existentes são pouco consensuais quanto à sua eficácia. A sua etiologia ainda é estudada, podendo estar relacionada com a infeção pelo Vírus do Papiloma Humano.

Phenotype and cell proliferation activity of duct-like structures in human sublingual glands: a histological and immunohistochemical study.

There are several age-related microscopic changes in the salivary glands, including the increase in the number of duct-like structures (DLS). However, the true origin and the phenotype of the DLS are not known. Objective To evaluate the phenotype and the cell proliferation index of the DLS of human sublingual glands.Material and Methods Sixty sublingual glands obtained from human cadavers were divided into two groups - 0-30 and 61-90 years old. The phenotype was estimated by immunostaining for cytokeratin 19 (CK 19) and the S-100 protein as well as by the presence of mucin and glycogen. The cell proliferation index was determined by the Ki-67 antibody. The histochemical techniques used periodic acid-Schiff (PAS) and Alcian Blue. In each captured microscopic field, the DLS were counted to establish a percentage for the staining profile. The statistical analysis was accomplished using Student’s t-test, the Mann-Whitney test and Pearson’s correlation coefficient (p<0.05).Results Comparing both groups, only CK 19 showed a statistically significant difference (p=0.033), with the strongest expression in the elderly group. There was no significant difference between PAS and Alcian Blue (p=0.270). In both groups, the immunostaining for CK 19 was stronger than that for S-100 (p=0.004;p<0.001), but there was no correlation between the two immunomarkers (ρ=-0.163; p=0.315). There was no immunostaining for Ki-67.Conclusions DLS demonstrate a ductal phenotypic profile and do not present cell proliferation activity. DLS may represent a regressive process arising from acini or represent the result of metaplasia.

Keratin Expression Patterns in Stromal Cells of Benign Sclerosing Lesions of the Breast: A Potential Diagnostic Pitfall.

Metaplastic carcinomas arising in association with benign sclerosing lesions (BSLs) are rare malignancies in which a neoplastic spindle cell proliferation can be recognized extending beyond the boundaries of the complex sclerosing lesion or papilloma. However, in cases in which the metaplastic carcinoma is of the low-grade fibromatosis-like type or is a low-grade adenosquamous carcinoma, distinction from the background BSL can be a significant challenge. Cytokeratin (CK) and/or p63 immunostains are helpful in confirming the diagnosis of metaplastic carcinoma, but the expression patterns of these markers in the stromal cells of BSLs have not been well characterized. To characterize the expression patterns of CKs and p63 in BSLs. We evaluated the spindle cell component of 55 BSLs using CK 5/6, CK 903, CK MNF116, and p63. A total of 45 cases (81%) showed no staining for CKs or p63 in benign stromal cells. CK 5/6, CK 903, and p63 were positive in one case each. CK MNF116 stained spindle cells within 10 BSLs. No cases showed spindle cell reactivity for all 4 markers. Positive cases demonstrated very focal, weak staining of spindle cells; only 1 case showed focal, moderate CK staining. Spindle cell positivity was not associated with lesion type, growth pattern, spindle cell atypia, or mitoses. These findings suggest that although the presence or absence of expression of CK 5/6, CK 903, and p63 may be useful to distinguish BSL from metaplastic carcinomas arising in this setting, CK MNF116 positivity may be a diagnostic pitfall.

Immunohistochemical and histopathological characters of renal mesenchymal tumor in a Wistar rat

A 15 weeks old male Wistar rat was having abdominal distention. Postmortem examination revealed a highly expansile mass with large cystic cavities and nodular growths on the abdominal wall. The tumor mass caused enlargement of the abdomen and nearly replaced the right kidney. The lesion was diagnosed as renal mesenchymal tumor by histomorphological examination which was aided by immunostaining. The distinctive feature of the present case was that the tumor was positive S100 and cytokeratin immunostaining, which has not been reported earlier.

Transitional cell carcinoma of urinary bladder with metastasis in lumbar vertebrae and spinal cord compression in an ocelot(Leopardus pardalis)

This paper reports a case of nonpapillary and infiltrative transitional cell carcinoma (TCC) of the urinary bladder with metastasis of lumbar vertebrae and spinal cord compression in an adult female ocelot (Leopardus pardalis), from the Mato Grosso state, Brazil. The ocelot had pelvic limb paralysis and skin ulcers in the posterior region of the body and was submitted to euthanasia procedure. At necropsy was observed a multilobulated and irregular shaped, yellowish to white nodule in the urinary bladder. The nodule had a soft consistency and arised from the mucosa of the urinary bladder extending throughout the muscular layers and the serosa. Nodules of similar appearance infiltrating the vertebral column the at L6 and L7 vertebrae with corresponding spinal canal invasion were also observed. The histological evaluation showed epithelial neoplastic proliferation in the urinary bladder with characteristics of nonpapillary and infiltrative TCC, with positive immunohistochemical staining for pancytokeratin, and strong immunostaining for cytokeratin of low molecular weight, and weak or absent labeling for high molecular weight cytokeratin. This is the first report of TCC of urinary bladder in ocelot in Brazil.

Atypical Fibroxanthoma of Scalp: A Paradoxical Benign Tumour.

Atypical fibroxanthoma (AFX) is a recently described soft tissue tumour by World Health Organization as a benign tumour of uncertain differentiation, occurring on sun exposed skin in elderly individual [1]. The bizarreness as well as marked atypia seen in the cells raises a first impression of malignant tumour, whereas it is rarely so. It has a male predominance (70%) with an average age of presentation of 71.9 years, ranging from 29–91 years [2]. An 81-year-old male presented with hard nodular scalp swelling since 7 to 8 years with a rapid size increase since 3 months. On examination there was a nodulo-ulcerative lesion measuring 5cm x 3cm on scalp with bleeding edges. He did not have any other cutaneous lesion or lymphadenopathy. Excisional biopsy done outside was reported as sebaceous carcinoma. The patient was referred to our hospital for further management. On review of biopsy slides, the tumour cells were spindled, plump to epithelioid with marked pleomorphism, arranged in haphazard, vaguely fascicular or storiform patterns [Table/Fig-1a]. Tumour cells had hyperchromatic and multilobulated bizarre nuclei with scattered multinucleate tumour giant cells. Some cells of the lesion contained vacuolated and lipid-containing cytoplasm similar to xanthoma raising doubts of sebaceous carcinoma, as diagnosed outside [Table/Fig-1b]. On extensive IHC work up, the tumour cells were positive for Vimentin (Dako;V9) [Table/Fig-1c], CD68 (Leica;514H12), CD10 (Dako;56C6) [Table/Fig-1d] and negative for Cytokeratin (Biogenix;AE1+AE3) [Table/Fig-1e], EMA (Dako, E29), SMA (Dako;1A4), S 100 (Dako;IS504), Desmin (Dako;D33), Caldesmon (Dako; H-CD) and HMB 45 (Dako;HMB45) [Table/Fig-1f]. A final diagnosis of atypical fibroxanthoma was made. [Table/Fig-1]: Histopathological examination showing large bizarre cells with abundant foamy cytoplasm and atypical mitosis (HE b) 400x). Immunohistochemistry showing positivity for Vimentin (c), CD10 (d) and negativity for Cytokeratin (e), HMB45 ... Making a diagnosis of atypical fibroxanthoma is challenging and the diagnosis should be made by exclusion of other differential diagnoses after applying the stringent histological criteria and a broad panel of immunostains. Cutaneous carcinomas and melanoma must be ruled out by immunohistochemistry. Absent immunostaining for cytokeratins, S100 and HMB45 in AFX are helpful for excluding sebaceous carcinoma along with squamous cell carcinoma and malignant melanoma. Other differentials are malignant fibrohistiocytoma, atypical Fibrous Histiocytoma /dermatofibroma with monster Cells and pleomorphic fibroma [Table/Fig-2]. By definition, in AFX tumour cells lack expression of S100, cytokeratin, CD34, desmin and h-caldesmon. Previously our case was misdiagnosed as sebaceous carcinoma due to pleomorphism, abundant clear to foamy cytoplasm and bizarre pleomorphic nuclei. CD10 is a useful marker for AFX and is positive in 95%–100% cases of AFX [2]. A histiocytic marker CD 68 is positive in more than half of cases [3]. AFX shows a very good prognosis after surgical excision [4]. Reports of metastasis are very rare, and recurrence is uncommon [5]. Distinction from high grade sarcoma is crucial to prevent inappropriate aggressive therapy. [Table/Fig-2]: Differential diagnoses of atypical fibroxanthoma

Immunohistochemical characterisation of a canine case of granular cell type trichoblastoma: a case report

Herein we describe a case of granular cell type trichoblastoma in a dog. A nine-year-old, intact male Scottish terrier presented with a solitary, exophytic and non-ulcerated nodule on the shoulder. Histopathologically, the nodule showed a ribbon-like structure, which was composed of branching cords of epithelial cells surrounded by fibrous stroma. In addition, islands of large clear cells were also noted. The clear cells showed eccentric nuclei and abundant cytoplasmic vacuoles, which were strongly positive for periodic acid-Schiff staining. Cytoplasmic immunostaining for cytokeratin (CK) 14 and nuclear immunostaining for p63 were observed in the neoplastic cells. In contrast, CK18 staining was scant. Furthermore, the hair bulge stem cell marker CK15 showed strong cytoplasmic staining. Based on these histopathological and immunohistochemical findings, the tumour was diagnosed as a granular cell type trichoblastoma, which is rarely reported in dogs. &amp;nbsp;

Tumor budding in colorectal carcinoma assessed by cytokeratin immunostaining and budding areas: possible involvement of c-Met.

Tumor budding/sprouting has been shown to be an independent adverse prognostic factor in T1 and T3N0 colorectal carcinomas, however, its assessment could be improved by more accurate identification of budding carcinoma cells and consideration of budding areas. Moreover, tumor budding mechanisms are yet to be defined. In this study, we evaluated the identification of budding tumor cells by either H&E staining alone or H&E with immunohistochemistry and developed a scoring system based on budding grades and areas. We examined whether the budding score correlated with clinicopathologic features and prognosis and the association between tumor budding/sprouting and c-Met protein expression and phosphorylation and MET gene copy numbers because c-Met is known to play an important role in colorectal carcinoma tumorigenesis. Cytokeratin immunohistochemistry could identify tumors with shorter disease-free survival (DFS) from the low-grade budding group assessed with H&E alone. High budding scores based on budding grade and area were more significantly correlated with DFS than scores obtained using the budding grade alone. In tumors with a high budding score, c-Met expression and phosphorylation levels and MET gene copy numbers were significantly increased at the invasive front compared with those in superficial tumor portions. This study showed for the first time that high levels of phospho-c-Met at the invasive front were significantly associated with a high budding score and shorter DFS. In conclusion, a budding score assessed by budding grades and budding-positive areas correlates highly with clinicopathologic aggressive features of colorectal carcinoma.

Serine protease inhibitor A3K suppressed the formation of ocular surface squamous metaplasia in a mouse model of experimental dry eye.

To investigate the effects and possible mechanisms of serine protease inhibitor A3K (SERPINA3K) on the formation of ocular surface squamous metaplasia in a mouse dry eye model induced by topical benzalkonium chloride (BAC). The eye drops containing SERPINA3K were topically administered during the induction of BAC-induced dry eye. The clinical indications of dry eye were evaluated on day (D)16, including tear break-up time (BUT), tear volume, corneal fluorescein staining, and inflammatory index. Global specimens were collected on D16 and the following examinations were performed: histologic investigation, immunostaining of cytokeratin 10 (K10), p63 and Ki67 in the cornea, and Western blot analysis of tumor necrosis factor-α (TNF-α). Serine protease inhibitor A3K suppressed the formation of BAC-induced dry eye, presenting with longer BUTs, lower corneal fluorescein staining scores, and inflammatory index, while no significant changes in tear volume. It also reduced the severity of abnormal differentiation and proliferation on ocular surface with lower expressions of K10, p63, and Ki67, and retained the number of goblet cells in the conjunctival fornix. Serine protease inhibitor A3K significantly decreased the levels of TNF-α in the cornea. Topical application of SERPINA3K ameliorated the severity of ocular surface squamous metaplasia and suppressed the formation of BAC-induced dry eye.

Concurrent loss of INI1, PBRM1, and BRM expression in epithelioid sarcoma: implications for the cocontributions of multiple SWI/SNF complex members to pathogenesis.

The loss of INI1 (SMARCB1) expression, caused by SMARCB1 (INI1, SNF5L4, BAF47) inactivation, frequently occurs in epithelioid sarcoma (ES) and could aid in confirming the diagnosis. Except for INI1, the expression of switch in mating type/sucrose nonfermentation complex members in ES has never been examined. In this study, the expression of key subunits of this complex-INI1, BRG1 (SMARCA4), BRM (SNF2L2, SMARCA2), PBRM1 (hPB1, BAF180), and BAF155 (SMARCC1)-was analyzed in 23 ES cases: 15 conventional and 8 proximal type. All of the cases were reviewed and reclassified by hematoxylin-eosin staining and immunostaining for cytokeratin AE1/3, epithelial membrane antigen, CD34, vimentin, and INI1 expression. Of the 23 ES cases, 19 (82.6%) showed a loss of PBRM1, and 18 (78.3%), a loss of INI1. In most cases (17, 73.9%), loss of INI1 and PBRM1 expression was observed. The pattern of PBRM1 expression was similar to that of INI1, that is, not correlated with changes in cellular morphology. The concurrent loss of BRM, PBRM1, and INI1expression was detected in 2 cases with pure rhabdoid tumor features. The frequent observation of concurrent loss of INI1 and PBRM1 suggests that certain switch in mating type/sucrose nonfermentation complex components might act synergistically in the pathogenesis of ES by unknown mechanisms and that these components could provide new targets for therapy. The usefulness of PBRM1 as a biomarker of ES and its mechanism in ES require further investigation. Loss of BRM in ES with pure rhabdoid features suggests that BRM might be involved in the underlying mechanisms of this type of ES.

Micrometastasis of hypopharyngeal cancer.

The aim of this study is to estimate the incidence and clinical impact of lymph node micrometastases in hypopharyngeal squamous cell cancer (HSCC). In this retrospective study, we enrolled 58 patients who have undergone surgery for HSCC (between January 2004 and January 2011). Pharyngolaryngectomy and oesophagectomy with selective bilateral neck dissection was performed in all patients. Based on standard histological examination, 17 patients met N0 and 8 patients met N1 criteria and were further evaluated for the presence of micrometastases and isolated tumour cells (ITC). Following immunohistochemical analysis, the patients were grouped according to the presence of micrometastases and ITCs. In the pN0 group, cytokeratin-positive cells were detected in five patients, and they were marked as N0/CK+. Among these five patients, two were found to harbour micrometastases and ITCs, whilst in three, only ITCs were found. Two patients (11.75 %) were upstaged to pN1. The patients marked as N0/CK+ had a statistically significant worse overall survival rates than pN0 patients with tissue samples read as negative for cytokeratin immunostaining (p = 0.019, p < 0.05). In the pN1 group, cytokeratin-positive cells were detected in two patients, with one patient showing micrometastases and ITC, and the other showing ITC only. One patient was upstaged to pN2. Patients with lymph node micrometastases and ITC had worse overall survival rates, which may indicate that more aggressive post-operative treatment regimens should be considered for these HSCC patients.

761 Zebrafish Studies Show That Add3, Not Xpnpep1, Is a Biliary Atresia Risk Gene

Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the intraand extrahepatic bile ducts of neonates. Although BA is the most common identifiable cause of obstructive jaundice in infants, and is the leading indication for pediatric liver transplantation, the etiology remains elusive. We investigated the importance of genes identified in genome-wide association studies (GWAS) of BA patients using zebrafish. The Matthews's lab has demonstrated the utility of the zebrafish system to test the functionality of genes identified in GWAS of BA by demonstrating that knockdown of gpc1 leads to biliary defects. A prior GWAS examined single-nucleotide polymorphisms in 200 Han Chinese BA patients and 481 ethnically matched controls. The strongest association was found for a region located between the XPNPEP1 and ADD3 genes on 10q24.2. Our genetic analysis confirmed the importance of this region in a separate cohort of patients. To determine whether loss of xpnpep1 and/or add3a leads to biliary defects we performed knockdown studies of the respective genes using morpholino antisense oligonucleotides (MO). We then examined their biliary function using the lipid reporter PED6, biliary development using cytokeratin immunostaining, and gene expression using quantitative PCR. We confirmed that xpnpep1 and add3a are expressed in the developing zebrafish liver. Knockdown of add3a led to decreased biliary function by PED6 screening, while xpnpep1 knockdown had only a mild effect. There were developmental biliary defects in the add3a morphants, as demonstrated by cytokeratin immunostaining. The transcription factor vhnf1, implicated in biliary development, was significantly downregulated in add3a morphants but not xpnpep1 morphants. add3a morphants also demonstrated increased expression of gli2a, a Hedgehog target, which is consistent with prior studies of BA patients and of zebrafish models of BA such as gpc1 knockdown. Interestingly, knockdown of both add3a and gpc1 resulted in a synergistic disruptive effect on biliary development. While GWAS identified ADD3 and XPNPEP1 as potential BA susceptibility genes, our results suggest that ADD3 is likely the more important gene in this regard. Like gpc1, add3a acts via Hedgehog signaling, supporting a role for this important pathway in BA pathogenesis.

762 Control of Microenvironmental Cues Enables the Differentiation of Pluripotent Stem Cell Derived Hepatocyte-Like Cells to an Adult Phenotype

Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the intraand extrahepatic bile ducts of neonates. Although BA is the most common identifiable cause of obstructive jaundice in infants, and is the leading indication for pediatric liver transplantation, the etiology remains elusive. We investigated the importance of genes identified in genome-wide association studies (GWAS) of BA patients using zebrafish. The Matthews's lab has demonstrated the utility of the zebrafish system to test the functionality of genes identified in GWAS of BA by demonstrating that knockdown of gpc1 leads to biliary defects. A prior GWAS examined single-nucleotide polymorphisms in 200 Han Chinese BA patients and 481 ethnically matched controls. The strongest association was found for a region located between the XPNPEP1 and ADD3 genes on 10q24.2. Our genetic analysis confirmed the importance of this region in a separate cohort of patients. To determine whether loss of xpnpep1 and/or add3a leads to biliary defects we performed knockdown studies of the respective genes using morpholino antisense oligonucleotides (MO). We then examined their biliary function using the lipid reporter PED6, biliary development using cytokeratin immunostaining, and gene expression using quantitative PCR. We confirmed that xpnpep1 and add3a are expressed in the developing zebrafish liver. Knockdown of add3a led to decreased biliary function by PED6 screening, while xpnpep1 knockdown had only a mild effect. There were developmental biliary defects in the add3a morphants, as demonstrated by cytokeratin immunostaining. The transcription factor vhnf1, implicated in biliary development, was significantly downregulated in add3a morphants but not xpnpep1 morphants. add3a morphants also demonstrated increased expression of gli2a, a Hedgehog target, which is consistent with prior studies of BA patients and of zebrafish models of BA such as gpc1 knockdown. Interestingly, knockdown of both add3a and gpc1 resulted in a synergistic disruptive effect on biliary development. While GWAS identified ADD3 and XPNPEP1 as potential BA susceptibility genes, our results suggest that ADD3 is likely the more important gene in this regard. Like gpc1, add3a acts via Hedgehog signaling, supporting a role for this important pathway in BA pathogenesis.

O56 SOFOSBUVIR/LEDIPASVIR WITH AND WITHOUT RIBAVIRIN FOR 8 WEEKS COMPARED TO SOFOSBUVIR/LEDIPASVIR FOR 12 WEEKS IN TREATMENT-NAÏVE NON-CIRRHOTIC GENOTYPE-1 HCV-INFECTED PATIENTS: THE PHASE 3 ION-3 STUDY

Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the intraand extrahepatic bile ducts of neonates. Although BA is the most common identifiable cause of obstructive jaundice in infants, and is the leading indication for pediatric liver transplantation, the etiology remains elusive. We investigated the importance of genes identified in genome-wide association studies (GWAS) of BA patients using zebrafish. The Matthews's lab has demonstrated the utility of the zebrafish system to test the functionality of genes identified in GWAS of BA by demonstrating that knockdown of gpc1 leads to biliary defects. A prior GWAS examined single-nucleotide polymorphisms in 200 Han Chinese BA patients and 481 ethnically matched controls. The strongest association was found for a region located between the XPNPEP1 and ADD3 genes on 10q24.2. Our genetic analysis confirmed the importance of this region in a separate cohort of patients. To determine whether loss of xpnpep1 and/or add3a leads to biliary defects we performed knockdown studies of the respective genes using morpholino antisense oligonucleotides (MO). We then examined their biliary function using the lipid reporter PED6, biliary development using cytokeratin immunostaining, and gene expression using quantitative PCR. We confirmed that xpnpep1 and add3a are expressed in the developing zebrafish liver. Knockdown of add3a led to decreased biliary function by PED6 screening, while xpnpep1 knockdown had only a mild effect. There were developmental biliary defects in the add3a morphants, as demonstrated by cytokeratin immunostaining. The transcription factor vhnf1, implicated in biliary development, was significantly downregulated in add3a morphants but not xpnpep1 morphants. add3a morphants also demonstrated increased expression of gli2a, a Hedgehog target, which is consistent with prior studies of BA patients and of zebrafish models of BA such as gpc1 knockdown. Interestingly, knockdown of both add3a and gpc1 resulted in a synergistic disruptive effect on biliary development. While GWAS identified ADD3 and XPNPEP1 as potential BA susceptibility genes, our results suggest that ADD3 is likely the more important gene in this regard. Like gpc1, add3a acts via Hedgehog signaling, supporting a role for this important pathway in BA pathogenesis.

P1163 ALL-ORAL THERAPY WITH DACLATASVIR IN COMBINATION WITH ASUNAPREVIR AND BMS-791325 FOR TREATMENT-NAIVE PATIENTS WITH CHRONIC HCV GENOTYPE 4 INFECTION

Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy affecting the intraand extrahepatic bile ducts of neonates. Although BA is the most common identifiable cause of obstructive jaundice in infants, and is the leading indication for pediatric liver transplantation, the etiology remains elusive. We investigated the importance of genes identified in genome-wide association studies (GWAS) of BA patients using zebrafish. The Matthews's lab has demonstrated the utility of the zebrafish system to test the functionality of genes identified in GWAS of BA by demonstrating that knockdown of gpc1 leads to biliary defects. A prior GWAS examined single-nucleotide polymorphisms in 200 Han Chinese BA patients and 481 ethnically matched controls. The strongest association was found for a region located between the XPNPEP1 and ADD3 genes on 10q24.2. Our genetic analysis confirmed the importance of this region in a separate cohort of patients. To determine whether loss of xpnpep1 and/or add3a leads to biliary defects we performed knockdown studies of the respective genes using morpholino antisense oligonucleotides (MO). We then examined their biliary function using the lipid reporter PED6, biliary development using cytokeratin immunostaining, and gene expression using quantitative PCR. We confirmed that xpnpep1 and add3a are expressed in the developing zebrafish liver. Knockdown of add3a led to decreased biliary function by PED6 screening, while xpnpep1 knockdown had only a mild effect. There were developmental biliary defects in the add3a morphants, as demonstrated by cytokeratin immunostaining. The transcription factor vhnf1, implicated in biliary development, was significantly downregulated in add3a morphants but not xpnpep1 morphants. add3a morphants also demonstrated increased expression of gli2a, a Hedgehog target, which is consistent with prior studies of BA patients and of zebrafish models of BA such as gpc1 knockdown. Interestingly, knockdown of both add3a and gpc1 resulted in a synergistic disruptive effect on biliary development. While GWAS identified ADD3 and XPNPEP1 as potential BA susceptibility genes, our results suggest that ADD3 is likely the more important gene in this regard. Like gpc1, add3a acts via Hedgehog signaling, supporting a role for this important pathway in BA pathogenesis.

Mammary diffuse fibroadenomatoid hyperplasia in water buffalo (Bubalus bubalis): three cases

The current report describes 3 rare cases of mammary diffuse fibroadenomatoid hyperplasia in water buffalo (Bubalus bubalis). All of the animals were between 10 and 12 months of age. Grossly, the lesions consisted of severe diffuse swelling with homogeneous large masses in the udder. Surgical removal of the masses was curative. Microscopically, there was severe hyperplasia of the mammary epithelium and numerous well-differentiated and mildly pleomorphic acini and their associated ducts. Moderate proliferation of the fibrous connective tissue and the myoepithelial cells near the proliferating acini was also evident. The hyperplastic epithelial cells exhibited positive immunostaining for cytokeratin, estrogen receptors, and progesterone receptors. In addition, the myoepithelial cells displayed moderate positivity for alpha smooth muscle actin. Based on the clinical, morphologic, and immunohistochemical findings, a diagnosis of mammary diffuse fibroadenomatoid hyperplasia with probable hormonal influence was made.

Prognostic Significance of Epithelial-Mesenchymal Transition of Extracapsular Spread Tumors in Lymph Node Metastases of Head and Neck Cancer

The extracapsular spread (ECS) of lymph node metastasis (LNM) reflects tumor aggressiveness and is associated with poor survival and risk of distant metastasis. In this study, we aimed to explore the prognostic significance of epithelial-mesenchymal transition (EMT) of ECS tumors in LNM of head and neck cancers. We collected LNM samples from head and neck cancer patients (follow-up >2years) and made 20 ECS(-): ECS(+) pairs (1:2) of LNM (N=60), matched by the primary sites and by T and N classifications. Immunostaining of cytokeratin, E-cadherin, vimentin, and CD31 were performed and quantified to determine the epithelial-mesenchymal transition percent (EMT%), defined as vimentin(+)/cytokeratin(+) area of ECS. Univariate and multivariable analyses of clinic-pathologic factors, including EMT% of ECS, were conducted to identify the significant prognosticators. In addition, the anatomical relationship between CD31 vessels and ECS tumors was analyzed. Rather than the presence of ECS in LNM, higher EMT% (>50%) of ECS strongly correlated with the worse overall and disease-free survival and had more frequent recurrence and distant dissemination in their clinical courses. ECS tumors intermingled closely with Ki-67(-) CD31(+) non-proliferating perinodal blood vessels. Particularly, vimentin(+) ECS areas exhibited a higher density of CD31(+) perinodal vessels than did vimentin(-) ECS. High EMT scores of ECS tumors in LNM predict an unfavorable prognosis and systemic dissemination more accurately than the simple presence of ECS in LNM in head and neck cancer patients.

Evidence from a Mouse Model That Epithelial Cell Migration and Mesenchymal-Epithelial Transition Contribute to Rapid Restoration of Uterine Tissue Integrity during Menstruation

BackgroundIn women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood.MethodologyA modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4) withdrawal; mice received a single injection of bromodeoxyuridine (BrdU) 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET) was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis.Principal FindingsMice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation.Conclusions/SignificanceThese studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and mesenchymal cell differentiation in restoration of an intact epithelial cell layer. These insights may inform development of new therapies to induce rapid healing in the endometrium and other tissues and offer hope to women who suffer from heavy menstrual bleeding.

Upper gastrointestinal cancers: Are all carcinomas truly carcinomas?

20 Background: Poorly differentiated cancer of the upper GI tract is a challenging diagnosis for the pathologist, who will rely on a positive cytokeratin immunostaining to confirm the epithelial origin of the tumor. Being confronted with a highly unusual case (a poorly differentiated esophageal “carcinoma” with immunophenotypic and genetic sarcoma-like features, arising in the background of high grade Barrett dysplasia), we retrospectively investigated whether other cancer cases displayed the same unusual features. Methods: 34 poorly differentiated GI cancers were selected based on availability of FFPE. All cases were investigated by (1) immunohistochemistry for selected carcinoma and sarcoma markers (prekeratin, NCAM, vimentin, CD99) and by (2) FISH for presence/absence of abnormalities of 2 genes, SYT and EWS, that are frequently rearranged in synovial and Ewing’s sarcoma respectively. Results: 6/34 cases displayed an aberrant immunophenotype, with expression of NCAM, vimentin, and/or CD99. FISH revealed a substantial number (9/34) of SYT and EWS related anomalies: 1 EWS rearrangements, 1 SYT rearrangements, 1 SYT and 1 EWS focal amplification, and 4 aneuploidies. While EWS and SYT are (mostly) fused to ERG or SSX1/2 in Ewing’s and synovial sarcoma respectively, these genes were found to be intact in our EWS and SYT rearranged cases. Conclusions: Our data show that SYT &amp; EWS abnormalities are recurrently occurring in poorly differentiated GI cancers that are (mis?)diagnosed as carcinomas. While EWS rearrangement has previously been described in cancer types other than Ewing’s sarcoma, SYT rearrangement was so far thought to be exclusive to the diagnosis of synovial sarcoma. In addition, EWS/SYT abnormalities in our series seemed to target cancers that are at risk of not responding well to the conventional chemoradiotherapy regimen. Of interest, the one “carcinoma” patient that started this study went into complete remission after being administered a sarcoma targeted therapy. We also infer from our results that good histologic assessment of GI cancers is essential for clinical trials aiming at targeted therapy, as not all carcinomas may present true carcinomas.

Post-chemotherapy bilateral limbal stem cell deficiency

We report an extremely rare case of bilateral total limbal stem cell deficiency (LSCD) induced by long-term systemic chemotherapy for squamous cell carcinoma of the lung, and the outcomes of bilateral keratolimbal allograft transplantation (KLAT) in such a case. A 34-year-old male patient had underlying disease of squamous cell carcinoma in the right upper lung with extensive mediastinum and lymph node metastasis and received a series of systemic chemotherapy, including a combination of vinorelbine and cisplatin and a combination of gemcitabine, cisplatin, and docetaxel. According to clinical manifestations of ocular surface disorder, pathology findings, and immunostaining of cytokeratin 13 (CK13), LSCD secondary to systemic chemotherapy was diagnosed. KLAT was performed in both eyes. Although graft rejection developed in the right eye, complete re-epithelialization with favorable visual acuity occurred in the left eye 3 years after limbal transplantation.